1. Field of the Invention
The present invention relates to continuous sample detection, and in particular, to the use of multiple cells for facilitating measurements.
2. Discussion of Related Art
The effects of a radioactive tracer introduced into an organism may in part be determined by removing tissue samples for analysis. A known method of detecting these radioactive tracers is to pass extracts, digests, and other solutions derived therefrom through a chromatography column to separate their constituents into various fractions, in the usual fashion. The eluate from the chromatography column can then be passed through a device for detecting the radioactivity.
Radioactivity detection is often practiced with a flow-through detector that continuously monitors the radiation from samples flowing through a cell. When the continuous flow to the cell is the eluate from a high performance liquid chromatography column (HPLC) large amounts of information can be obtained through numerical and graphical analysis.
In a known detection technique, the eluate is continuously mixed with a scintillating solution and passed through a transparent tubular cell mounted in a sample block inside a light-tight box. Alternatively, scintillation solution need not be used and instead, the eluate is passed over insoluble scintillator particles within the cell. The faint light coming from the tubular cell is detected by a pair of photomultiplier tubes on opposite sides of the cell. See for example, U.S. Pat. No. 4,194,117.
There are occasions when the type of cell ought to be changed in systems of the above type. For example, two technicians might be sharing one detector system and each could require or prefer a different sample cell configuration. In that case, each technician would need to replace the sample cell before beginning test operations. Also, cell changes may be desirable when technicians use the detector with automatic sample injectors which can have a capacity of a hundred samples or more. Different batches of samples (or simply different preferences) may indicate the need for a different sample cell. In such an automated system, there is no provision for dealing automatically with changed requirements when proceeding to a new batch. Operators have therefore, settled with a compromise cell that is not ideally tailored to either batch.
Any attempt to run large batches can be frustrated if successive batches require a different sample cell and a compromise cell is unsatisfactory. In such situations, the batches cannot be run automatically, but the process must stop so that an operator can intervene and change the sample cell.
Another area of unfortunate compromise results from the fact that the amount of radiation from a sample will change over time as different fractions are delivered in the eluate from a chromatography column. It is difficult to choose a sample cell that will work adequately for low activity peaks as well as high activity peaks occurring in one sample. Low activity peaks can be more easily measured with larger cell volumes that have greater sensitivity. On the other hand, with large cell volumes and increased counting time, it is possible to overwhelm the electronics with excessive numbers of counts. Also the larger cell volumes have less resolution and may not be able to distinguish between closely spaced peaks. Accordingly, operators will try to strike a balance between these competing requirements.
Changing from one flow-cell to another flow-cell is difficult. In most instruments, the cover must be removed, the high voltage disabled, plumbing fittings disconnected, the cell removed (potentially causing problems with ambient light as explained below), the new cell installed, the cover replaced, and the high voltage reestablished. Finally, the measurement can be made. To later return to the original cell, all of the forgoing steps must be repeated.
The high voltage must be turned off each time a cell is removed or inserted. Exposure of a photomultiplier to ambient light with the high voltage applied can cause irreparable damage, since the consequential high current flows will likely "strip" the photocathodes.
Regardless, removing the high voltage is inherently problematical, because photomultipliers are most quiet (i.e., least electronic noise) when kept at constant high voltage for long periods. Similarly, even with the high voltage off, exposure to ambient light can cause photocathodes to become light activated, requiring then a lengthy period of dark adaptation to reduce background noise to minimum levels.
Similarly, removal and ambient illumination of cells packed with solid scintillators (particularly the popular yttrium silicate) cause phosphorescence, again requiring a lengthy period of dark adaptation before the cell reaches its lowest backgrounds.
Furthermore, repetitively disconnecting and reconnecting fittings is an invitation to leaks, especially at the high pressures that are apt to be encountered in HPLC. Cleanup of leakage of radioactive solutions may have heavy consequences.
External standardization has been used in liquid scintillation systems to determine the quality of individual samples, especially for quenching phenomena. With that technique, an external radioactive source is brought near the sample to determine how the scintillation process varies in response to known radioactive stimulation. See for example, U.S. Pat. Nos. 3,609,361; 3,188,468; and 3,381,130. For other quenching correction techniques, see U.S. Pat. Nos. 4,008,393 and 4,292,520. See also U.S. Pat. Nos. 3,935,449 and 4,967,048.
In U.S. Pat. No. 5,559,324 standardization is performed on a continuous flow, radiochromatography system. That disclosed device has a sample holder mounted on an axially movable block. A standard holder is mounted on the rear of the cell block. Accordingly, the sample cell and standards can be moved to place either one in the proximity of a photomultiplier tube. Thus, the system can be calibrated, for example, with a sealed standard without the need to disconnect the sample cell from the sample lines. This reference teaches the advantage of avoiding exposing the photomultiplier to ambient light and avoiding the inconvenience of disconnecting and reconnecting plumbing to the sample cell. The reference, however, does not teach the importance of quickly changing flow sample cells.
In U.S. Pat. No. 4,853,945 multiple cells are used simultaneously, not sequentially. FIG. 5 shows eluate from a chromatography column flowing past six (6) stations. Each of these stations are connected by light pipes to separate photomultipliers. While there are effectively different stations at which measurements are made, this system has the disadvantage of requiring simultaneous measurement. The system is designed to increase the effectiveness of the photomultipliers by monitoring six (6) stations with four (4) tubes. The origin of pulses is determined by correlating the pulses. Furthermore, systems relying on correlation do not operate accurately, because accidental coincidences occur once the true count rate in one sample or cell becomes greatly elevated, as is often the case. Even if there are no true counts in other samples or cells being examined at the same time, there will be a high rate of accidental coincidences arising from these photomultiplier pairs that involve the hot sample and the others.
The nature of these false coincidences can be also understood by referring to U.S. Pat. No. 3,723,736 which shows a trio of equiangularly spaced photomultiplier tubes. Each of three samples is positioned among the photomultiplier tubes to straddle and shine upon a pair of the tubes. Assume a first sample has a very high count rate, a second sample has nothing, and the third sample has some modest count rate. The coincidence rate from the photomultiplier pair illuminated by the very high count rate will likely give a reasonably accurate result for the high count rate. Still, one member of this highly stimulated pair, however, will also be correlated with the remaining tube to determine the count rate for the inactive sample. There will be accidental coincidences for the pair covering the inactive sample, since one of the pair is highly stimulated (plus noise) and the other will be modestly stimulated but not by the inactive sample. Thus, the inactive sample will seem to have correlated counts from its two tubes although they will be derived mostly from accidental correlation between the other two samples (plus noise), one of which is highly active. Further, if any of these samples should have luminescence problems, a common occurrence, there will be some effect on the other samples.
The accidental coincidence rate of two (2) random noise generators is a function of n.sub.1, n.sub.2, and T, where n.sub.1 and n.sub.2 are the non-coincident count rates of each tube and T is the coincidence resolving time of the circuit. Because a product is involved, when either count rate goes up substantially, the accidental rate climbs correspondingly. One might conclude that this could be made negligible with faster coincidence circuitry, but an unfortunate characteristic of photomultipliers is that transit time (the time from propagation of a pulse at the photocathode until it can be sensed at the anode) is rather variable. If the coincidence time is made too short, true coincidences are missed and the counting efficiency goes down. U.S. Pat. No. 4,853,945 acknowledges this problem (column 4, lines 18, et seq.).
Another disadvantage with systems of this type is that as the sample moves along through the various cells, it is measured by four (4) different pairs of photomultipliers, each possibly having slightly different responses. Since the different cells will be in principle working together and simultaneously, it is unclear that such a system would work properly if the cells had different characteristics.
Another system shown in U.S. Pat. No. 4,027,163 shows a pair of serially connected radioactivity detectors. The downstream detector also has a standard radioactive source. This reference is designed to collect data from the two (2) collectors simultaneously and, therefore, is not designed to avoid the expense of having multiple detectors for multiple sites. U.S. Pat. Nos. 4,924,093 and 5,416,329 show detection of radiation from discrete samples, that is, vials. These references are unconcerned with systems for monitoring radiation from a flowing sample.
See also U.S. Pat. Nos. 4,791,820; 4,495,420; 4,528,450; and 5,328,662.
Accordingly, there is a need for an improved, continuous sample detector that provides the benefit of the specialized characteristics offered by different types of sample cells, but without multiplying the time or the amount of sensors needed to achieve such flexibility and adaptability.